This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Several proteomic studies of mammalian and yeast mitochondria have identified a growing list of over 1000 proteins in the mitochondrial proteome. The overlap between various studies is limited to 60-80% and the relative abundance of these components varies with tissue type. The number of mitochondrial proteins has been estimated at 2000, based on an endosymbiont model for mitochondrial evolution (Gabaldon, 2004), thus the mitochondrial proteome is still incomplete. The vast majority of mitochondrial proteins are nuclear encoded and are expected to contain N-terminal peptide signals used for mitochondrial targeting. Interestingly, only one third of the proteins in the mammalian mitochondrial proteome are predicted to contain a signal peptide motif using bioinformatic analysis, while another third are easily annotated with known mitochondrial enzymatic functions. The remaining third of the sample are typically novel mitochondrial localized proteins of ambiguous function, and in some studies this number is greater than half of the total protein list. Conspicuously absent from many of these protein lists are the apoptosis regulating proteins, which associate with mitochondrial membranes in a highly regulated pathway for cell death. This project will address 1) functional annotation of mitochondrial proteins using controlled biology and pharmacological induction of early apoptosis signaling and 2) analysis of N-terminal peptide for targeting of proteins to mitochondria. To accomplish these goals, we are investigating free flow electrophoresis (FFE) purification of intact mitochondria, reducing contamination by high abundance ribosome and nuclear components. We are also using a chemical biology enzymatic approach to label with subtiligase the free N-termini of mitochondrial proteins for affinity capture. The role of the Mass Spectrometry Facility in this project is to handle all aspects of the organelle preparation, peptide sequencing and bioinformatic analysis of N-terminal peptides.